A genetically encoded reporter platform to dissect the O-glycoproteome

每一个活细胞的表面都有一层保护性的糖分子，叫做糖萼。这些分子比膳食糖复杂得多，是生命的重要组成部分。作为细胞的最外层，糖萼通常是与其他细胞、病原体（如病毒）和信号分子相互作用的第一部分。糖分子的结构是高度可变的，微小的变化可以产生深远的影响，例如对新陈代谢和建立有效的免疫反应。与其他生物分子不同，糖不直接编码在基因组中-没有DNA模板来编码它们。相反，被称为酶的分子机器从简单的构建模块构建复杂的糖。这些酶形成了一条装配线，依次将每个构建模块整合到蛋白质中，从而产生了巨大的糖结构复杂性。酶之间的相互作用决定了最终形成的结构。为了了解糖分子在健康和疾病中的作用，了解酶的功能是很重要的。我们的重点是一类称为GalNAc-Ts的酶，它将某种称为GalNAc的糖构建块整合到蛋白质中。这些蛋白质最终会出现在细胞表面或血液中，并影响许多不同的过程。当GalNAc-Ts不能正常工作时，会出现一系列严重的影响。例如，癌细胞通常有太多的GalNAc-T酶。由于细胞中有许多不同的GalNAc-T，它们的作用略有不同，因此很难在分子水平上理解它们如何一起工作。这种理解是重要的，因为它将阐明生物学中一些最基本的过程，并为新药的设计提供线索。为了了解GalNAc-Ts如何协同工作，我们将开发所谓的报告试剂。如果这些试剂对某种GalNAc-T酶具有特异性，它们应该告诉我们GalNAc-T作用于哪个靶蛋白，以将糖GalNAc转移到哪个靶蛋白上。为了使试剂具有特异性，我们将使用一个技巧：试剂被设计为仅由单个GalNAc-T使用，该GalNAc-T经过轻微改变或改造。因为没有一个正常的，不变的GalNAc-T可以结合试剂，他们不会给我们一个信号。然后我们可以使用这些报告试剂告诉我们哪个GalNAc-T作用于哪个蛋白质。我们将使用这种技术来研究整个GalNAc-T家族。我们将建立一个特定试剂的平台，并生成与科学界共享的数据。我们的方法将为我们提供重要的洞察糖被纳入活细胞的方式。这些研究将为基础和应用研究的许多不同方面铺平道路，从了解生理学的分子机制到产生药物。

Every single living cell carries on its surface a protective layer of sugar molecules called the glycocalyx. These molecules are much more complex than dietary sugar and are an important component of life. As the outermost layer of cells, the glycocalyx is often the first part that interacts with other cells, pathogens such as viruses, and signalling molecules. The structure of the sugar molecules is highly variable, and small changes can have a profound impact, for example on metabolism and on mounting an effective immune response.Unlike other biomolecules, sugars are not directly encoded in the genome - there is no DNA template that codes for them. Instead, molecular machines called enzymes build complex sugars from simple building blocks. These enzymes form an assembly line that sequentially incorporates each building block into proteins, creating a huge complexity of sugar structures. The interplay between enzymes determines which structures are eventually made. In order to understand the roles of sugar molecules in health and disease, it is thus important to understand how enzymes function.Our focus is on a large class of enzymes called GalNAc-Ts that incorporate a certain sugar building block called GalNAc into proteins. These proteins eventually end up on the cell surface or in the bloodstream and impact lots of different processes. When GalNAc-Ts do not function properly, a range of severe effects are seen. For instance, cancer cells often have too many GalNAc-T enzymes. As there are many different GalNAc-Ts with slightly different roles in a cell, it is very difficult to understand on a molecular level how they work together. This understanding is important as it will shed light on some of the most fundamental processes in biology and give clues about the design of new drugs.To understand how GalNAc-Ts work together, we will develop reagents that act as so-called reporters. If these reagents are specific for a certain GalNAc-T enzyme, they should tell us which target protein the GalNAc-T worked on to transfer the sugar GalNAc to. In order to make the reagents specific, we will use a trick: the reagents are designed such that they are only used by a single GalNAc-T that has been slightly altered or engineered. Since none of the normal, unchanged GalNAc-Ts can bind the reagents, they won't give us a signal. We can then use these reporter reagents to tell us which GalNAc-T worked on which protein.We will use this technique to study the entire GalNAc-T family. We will set up a platform of specific reagents, and generate data that will be shared with the scientific community. Our approach will give us important insight into the way sugars are incorporated into living cells. These studies will pave the way to many different aspects of basic and applied research, from understanding molecular mechanisms of physiology to generating drugs.